PROJECT SUMMARY/ABSTRACT Extracellular peptidases are critical regulators of endogenous opioid signaling. These proteolytic enzymes degrade enkephalins and other endogenous opioid peptides after secretion, thereby limiting the duration and magnitude of opioid receptor activation. Angiotensin-converting enzyme (ACE) degrades enkephalins and is expressed in the brain, with uniquely high expression in the striatonigral pathway formed by Drd1a medium spiny neurons (D1-MSNs). Our long-term objective is to understand circuit-specific regulation of endogenous opioid signaling by ACE in D1-MSNs. Preliminary show that pharmacological inhibition of ACE reduces excitatory synaptic transmission onto D1-MSNs in the nucleus accumbens. This effect involves endogenous opioid signaling, as it is blocked by opioid receptor antagonism with naloxone. However, ACE inhibition does not affect excitatory synaptic transmission onto Drd2 medium spiny neurons (D2-MSNs). The goal of this proposal is to identify the endogenous opioid peptide normally degraded by ACE, which thus mediates the physiological effect of ACE inhibition on D1-MSNs. While ACE is not primarily responsible for degrading conventional Met- or Leu- enkephalin, it can efficiently degrade a more exotic enkephalin congener: Met-enkephalin-Arg-Phe (MERF). This proenkephalin gene product is highly expressed in the nucleus accumbens and has unique properties, including high binding affinity for multiple opioid receptor subtypes and strong analgesic activity. Our central hypothesis is that ACE inhibition selectively prevents the degradation of MERF, causing depression of excitatory synaptic transmission onto D1-MSNs. In Aim 1, we will define the specificity with which ACE degrades MERF in nucleus accumbens tissue, using a combination of electrophysiology and liquid chromatography-tandem mass spectrometry. We predict that ACE inhibition will regulate MERF but not conventional enkephalins. In Aim 2, we will determine if MERF is responsible for the physiological effect of ACE inhibition in the nucleus accumbens, by generating a novel transgenic mouse that permits selective genetic disruption of MERF without altering other enkephalins. In the absence of MERF, we predict that ACE inhibition will not affect excitatory synaptic transmission onto D1-MSNs. We expect our conditional MERF knockout mouse will prove broadly useful for understanding other functions of this exotic enkephalin congener, which remains poorly understood. The selective effect of ACE inhibition on nucleus accumbens D1-MSNs is also striking, because excessive activation of these cells is associated with addiction-related behavior and other pathological states. Brain ACE may thus represent a novel target for circuit-specific pharmacotherapy to treat substance use disorders.